Previous studies have shown that HLA-G, a class Ib antigen, is produced in placentas and is present in high concentrations at the maternal-fetal interface HLA-G mRNA is processed into several different transcripts, two of which encode soluble isoforms. Experiments done in our laboratory established that soluble isoforms are not only present at the maternal-fetal interface but also circulate in maternal blood. Leukocytes at the maternal-fetal interface as well as blood leukocytes exhibit putative receptors for HLA-G, i.e., ILT2 and ILT4. To investigate the functional implications of these unique observations, we have developed recombinant soluble HLA-G1 (rsG1) and rsG2 in human embryonic kidney (HEK293) cells. Preliminary studies where human blood mononuclear cells were treated with rsG1 or rsG2 then analyzed using DNA microarrays showed that these proteins are biologically active. Each reproducibly altered a spectrum of mRNAs associated with cell viability, cell growth and differentiation, chemotaxis and migration, and production of certain cytokines and their receptors. In this application we propose in depth investigation of the sources, target cells and functional consequences of binding sHLA-G as well as studies aimed at establishing and primate model for studying HLA-G functions in vivo. Four specific Aims are planned. In Aim 1, the goal is to establish cellular sites of synthesis of sG1 and sG2. In Aim 2, we propose to determine target cells for rsG1 and rsG2 and establish the identity of rsG1 and rsG2 on (a) antigen presentation, (b) cytokine and cytokine receptor expression, (c) cell death pathways, and (d) chemotaxis and cell migration in resting and activated blood leukocytes. Because critical experiments to evaluate the effects of sHLA-G on pregnancy cannot be conducted in women, we are developing a baboon model. Six transcripts that are unique to the placenta and closely resemble the spectrum of HLA-G mRNAs have been isolated and sequenced. The goal of Aim 4 is to investigate production of proteins from these messages and the tissue distribution of the proteins. We expect the results of these studies to shed considerable light on the functions of sHLA-G and eventually to provide mechanistic explanations for the failure of some women to achieve successful pregnancy.